Plant extracts and alkaloids having antitussive activity

ABSTRACT

The present invention provides plant extracts, isolated alkaloids, synthetic alkaloids and compositions having antitussive activity. In some preferred embodiments, the plant extracts and the isolated alkaloids are from a plant of a genus in the family  Stemonaceae.  In other preferred embodiments, the plant extracts and the isolated alkaloids are from a plant of the genus  Stemona, Croomia,  or  Stichoneuron.  In especially preferred embodiments, the plant extracts and the isolated alkaloids are from the plant  Stemona tuberosa.  The present invention further provides methods for isolating such plant extracts and alkaloids. In addition, the present invention provides methods for reducing or suppressing coughing by administering plant extracts, alkaloids and compositions having antitussive activity.

FIELD OF THE INVENTION

This invention pertains to the field of plant extracts, isolated plantalkaloids, and synthetic alkaloids that have antitussive activity andwhich are useful as pharmaceutical agents for reducing or suppressingcoughing.

BACKGROUND OF THE INVENTION

Coughing provides a means for clearing the tracheal and bronchial treesof a accumulated secretions and/or foreign bodies. The mechanism ofcoughing is initiated by an appropriate stimulus which elicits a deepinspiration, followed by closure of the epiglottis and relaxation of thediaphragm. Thereafter, a sharp muscle contraction against the closedepiglottis occurs, thereby, producing increased pressure in theintra-thoracic and intra-airway passages. The positive intra-thoracicpressure causes a narrowing of the trachea due to enfolding of itscompliant posterior membrane and opens the epiglottis. When theepiglottis opens, the combination of the large pressure differentialbetween the thoracic cavity and the atmosphere, coupled with thenarrowing of the trachea, produces a massively increased rate of airflow through the trachea. The force created by this increased rate ofair flow can effect the clearance of expectorate mucus and foreignmaterials from the airway.

Coughing is caused by a variety of stimuli, including physiological,mechanical, or chemical stimuli. For example, coughing is produced byinflammatory mechanisms, mechanical disorders, and chemical and thermalstimulation. Also for example, inflammatory stimuli can be initiated byedema of the mucosal membranes. The edema, in turn, can be from anyetiology, for example, bacterial or viral infection, the common cold, orexcessive cigarette smoking. Inflammatory stimuli may also be elicitedby irritation from exudative processes such as post-nasal drip andgastric reflux. Such stimuli may arise in the airways, for example as inlaryngitis, bronchitis, pneumonia or an abscess in the lungs.

Further, mechanical stimuli, for example the inhalation of particulatematter, can cause coughing. Other mechanical disorders which result incompression of the air passages or increased pressure upon any area ofthe respiratory system may result in coughing. Such mechanicaldifficulties may arise from intra-mural or extra-mural etiologies. Forexample, extra-mural causes of coughing include extra-mural pressurecaused by an aortic aneurysm, granulomas, pulmonary neoplasms,mediastinal tumors, and the like. Intra-mural lesions, such asbronchiogenic carcinoma, bronchial adenoma, the presence of foreignbodies or bronchial asthma also result in coughing. Decreased pliancy ofthe respiratory membranes may also result in chronic coughing, as in thecase of patients suffering from cystic fibrosis.

Chemical stimuli, for example the inhalation of irritant gases (e.g.,cigarette smoke or chemical fumes) may also elicit coughing. Otherchemical entities result in coughing due to their reactive effect uponthe respiratory system itself or on the balance and uptake ofrespiratory gases. Additionally, many chemical agents induce coughingdue to their reaction with enzymes involved in the respiratory process.Lastly, thermal stimuli, such as the inhalation of either very hot orcold air, may also result in coughing.

In some disease states, a persistent cough can be the only or primarysymptom. For example, patients suffering from bronchial asthma canresult in incessant coughing. Moreover, in some medical conditions, forexample, asthma, the cough mechanism itself may further aggravate thepatient's condition. Asthma is a condition marked by recurrent attacksof paroxysmal dyspnea with wheezing, which is due to spasmodiccontraction of the bronchi. The condition is caused by variousetiologies. In some cases, asthma is the result of an allergic reaction.A variety of factors including vigorous exercise, chemical orparticulate irritation, or even psychological stress can stimulate orprovoke coughing. Moreover, the violent contractions of the thoraciccavity which accompany coughing further aggravates already irritatedrespiratory membranes. A review of the physiology of coughing ispresented by Karlsson et al., Pulmonary Pharmacology and Therapeutics(1999) 12:215-238.

A variety of antitussive drugs have been developed for the treatment ofcoughing, for example, morphine-like compounds and compounds that act onopioid receptors. However, these compounds have adverse side effects.For example, the use of morphine-like compounds are known to result inaddiction, respiratory suppression, and inhibitory action of smoothmuscle contraction (e.g., resulting in constipation), andpsychotomimetics. In particular, codeine is known to be highly addictiveand dextromethorphan is known to induce hallucinations, delusions, orother symptoms of a psychosis. Moreover, drugs having strong antitussiveactivity, for example codeine and dextromethorphan, are known toadversely act on the central nervous system. Further, drugs that act onopioid receptors are known to adversely effect urination (Leander et al.Pharmacol. Exp. Ther., 227:35 (1983). A review of antitussive drugs ispresented by Hey et al., Annual Reports in Medicinal Chemistry 35:53-62(2000) and Bolser, Pulmonary Pharmacology 9:357-364 (1996).

A variety of drugs are available for the treatment of coughing. However,the number of safe and effective antitussive agents devoid of unwantedside effects, for example, sedation and addiction, is limited. In viewof the serious adverse side effects of the drugs used to treat coughing,there is a need for antitussive drugs that are free of such side effectsand are effective in reducing or suppressing coughing. The presentinvention fulfills these and other needs.

SUMMARY OF THE INVENTION

The present invention provides, inter alia, antitussive compounds thatare free of side-effects and are effective in reducing or suppressingcoughing. In one aspect, the antitussive compounds are plant extracts ortheir derivatives such as from the family Stemonaceae.

As such, in one embodiment, the present invention provides a compoundhaving Formula I:

In Formula I, R¹ is selected from a hydrogen and anα(S)-methyl-γ(S)-butyroylactonyl; R² is selected from β-orientedhydrogen and an α-oriented hydrogen; R³ is selected from a β-orientedhydrogen and an α-oriented hydrogen; R⁴ is hydroxyl; R⁵ is selected froma hydroxymethyl and a carboxyl. In an alternative embodiment, R⁴ and R⁵together with the carbons to which they are attached, join to form asubstituted γ(S)-butyroylactonyl or substituted furane ring; and R⁶ isselected from a β-oriented hydrogen and an α-oriented hydrogen. Inanother embodiment, R⁴ and R⁵ together with the carbons to which theyare attached, join to form a substituted γ(S)-butyroylactonyl and R² andR⁶ are both absent and form a pyrrole ring, provided however, that whenR¹ is α(S)-methyl-γ(R)-butyroylactonyl, R³ is an α-oriented hydrogen.

In another embodiment, the present invention provides a pharmaceuticalcomposition, the composition comprising a compound having Formula I:

R¹ is selected from a hydrogen and an α(S)-methyl-γ(S)-butyroylactonyl;R² is selected from a β-oriented hydrogen and an α-oriented hydrogen; R³is selected from a β-oriented hydrogen and an α-oriented hydrogen; R⁴ ishydroxyl; R⁵ is selected from a hydroxymethyl and a carboxyl, oralternatively, R⁴ and R⁵ together with the carbons to which they areattached, join to form a substituted γ(S)-butyroylactonyl or substitutedfurane ring; and R⁶ is selected from a β-oriented hydrogen and anα-oriented hydrogen. In another embodiment, R¹ and R⁵ together with thecarbons to which they are attached, join to form a substitutedγ(S)-butyroylactonyl and R² and R⁶ are both absent and form a pyrrolering, provided however, that when R¹ isα(S)-methyl-γ(R)-butyroylactonyl, R³ is an α-oriented hydrogen, and apharmaceutically acceptable carrier.

In another embodiment, the present invention provides a method forreducing coughing in a subject, the method comprising: administering apharmaceutically effective amount of a compound having Formula I:

wherein: R¹ is selected from a hydrogen and anα(S)-methyl-γ(S)-butyroylactonyl; R² is selected from a β-orientedhydrogen and an α-oriented hydrogen; R³ is selected from β-orientedhydrogen and an α-oriented hydrogen; R⁴ is hydroxyl; R⁵ is selected froma hydroxymethyl and a carboxyl. In an alternative embodiment, R⁴ and R⁵together with the carbons to which they are attached, join to form asubstituted γ(S)-butyroylactonyl or substituted furane ring; and R⁶ isselected from a β-oriented hydrogen and an α-oriented hydrogen. Inanother embodiment, R⁴ and R⁵ together with the carbons to which theyare attached, join to form a substituted γ(S)-butyroylactonyl and R² andR⁶ are both absent and form a pyrrole ring, provided however, that whenR¹ is α(S)-methyl-γ(R)-butyroylactonyl, R³ is an α-oriented hydrogen,thereby reducing coughing in a subject.

In yet another embodiment, the present invention provides a Stemonaceaefamily plant extract having antitussive activity, wherein theStemonaceae family plant extract inhibits a cough in a subject. Incertain aspects, the genus belonging to the Stemonaceae family isselected from Stemona, Croomia, or Stichoneuron. Preferably, the speciesof the Stemona or Croomia genus is selected from S. collinsae, S.japonica, S. mairei, S. parviflora, S. sessilifolia, S. tuberosa, C.japonica, and C. heterosepala wherein C. represents Croomia. In onepreferred embodiment, the Stemona species is Stemona tuberosa. The plantextract can be an aqueous extract or a total alkaloid extract.

In other embodiments, the present invention provides a pharmaceuticalcomposition, comprising: a Stemonaceae family plant extract havingantitussive activity, wherein the Stemonaceae family plant extractinhibits a cough in a subject.

In still yet another embodiment, the present invention provides a methodfor reducing coughing in a subject, comprising: administering apharmaceutically effective amount of a Stemonaceae family plant extracthaving antitussive activity, wherein the Stemonaceae family plantextract inhibits a cough in a subject.

In yet another embodiment, the present invention provides a method forpreparing a total alkaloid extract having antitussive activity from aStemonaceae family plant, the method comprising: (a) contacting aStemonaceae family plant sample with an alcohol to form a liquor; (b)evaporating the alcohol in the liquor to form a syrup; (c) adjusting thesyrup to an acid pH to form a supernatant fraction; (d) adjusting thesupernatant fraction to a basic pH, and then extracting with an organicsolvent to form an organic solution; and (e) evaporating the organicsolvent to dryness to form a total alkaloid extract having antitussiveactivity.

In still yet another embodiment, the present invention further providesa method for preparing four Stemona alkaloids having antitussiveactivities from a total alkaloid extract as mentioned above, the methodfurther comprising separating, purifying and crystallizing the totalalkaloid extract, to form four Stemona alkaloids having antitussiveactivity. The four Stemona alkaloids can be separated using conventionalchromatographic techniques.

In still yet another embodiment, the present invention provides a methodfor preparing an aqueous extract having antitussive activity from aStemonaceae family plant, the method comprising: (a) contacting aStemonaceae family plant sample with an aqueous solvent to form aliquor; and (b) evaporating the liquor to dryness to form an aqueousextract having antitussive activity.

These and other embodiments will become more apparent when read with thedetailed description and drawings which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structural formulae of eight related Stemonaalkaloids. FIG. 1A shows the chemical structural formulae of compoundsB, C, D and E; FIG. 1B shows the chemical structural formulae of threesynthetic plant alkaloids, compounds F, G, and H; and FIG. 1C shows thechemical structural formula of the isolated plant alkaloid,neotuberostemonine (compound A).

FIG. 2 illustrates the antitussive effects of the aqueous extractderived from Stemona tuberosa. FIG. 2A shows the percentage of coughepisodes of the control; and FIG. 2B shows the ratio of cough latencybetween the extract and the control. The number of animals tested isindicated in the parenthesis. *P<0.05 compared with the vehicle control.

FIG. 3 illustrates the antitussive effects of the total alkaloid extractderived from Stemona tuberosa. FIG. 3A shows the percentage of coughepisodes of the control; and FIG. 3B shows the ratio of cough latencybetween the extract and the control. The number of animals tested isindicated in the parenthesis. **P<0.01 compared with the vehiclecontrol.

FIG. 4 illustrates the antitussive effects of neotuberostemonine(compound A). FIG. 4A shows the percentage cough episode of the control;and FIG. 4B shows the ratio of cough latency between compound A and thecontrol. The number of animals tested is indicated in the parenthesis.*P<0.05, **P<0.01, and ***P<0.001 compared with the vehicle control.

FIG. 5 illustrates the antitussive effects of compounds A, B, C, and Din animals. FIG. 5A shows the effects of compounds A, B, C, and Dadministered intraperitoneally (133 μmol/kg); and FIG. 5B shows theantitussive effects of compounds A and E administered orally (400μmol/kg). The number of animals tested is indicated in the parenthesis.*P<0.05 and ***P<0.001 compared with the corresponding vehicle control.Normal saline was used as the vehicle for compounds C and A, and 5%Tween 80 in normal saline was used as the vehicle for compounds B and D,administered intraperitoneally; whereas, 5% Tween 80 in normal salinewas used as the vehicle for compounds E and A, administered orally.

FIG. 6 illustrates the antitussive effects of compounds D, F, G and Hadministered intraperitoneally (133 μmol/kg). The number of animalstested is indicated in the parenthesis. *P<0.05 compared with thecorresponding vehicle control. 5% Tween 80 in normal saline was used asthe vehicle for compounds F, D and G, and normal saline was used as thevehicle for compound H.

FIG. 7 illustrates the effect of naloxone (1 mg/kg) on the antitussiveactivity produced by neotuberostemonine (compound A, 100 mg/kg) andcodeine (30 mg/kg). The number of animals tested is indicated in theparenthesis. **P<0.01 and ***P<0.001 compared with the vehicle control;and ##P<0.01 compared with the codeine treated group.

FIG. 8 shows the molecular structure of compound A.

FIG. 9 shows the molecular structure of compound B.

FIG. 10 shows the molecular structure of compound C.

FIG. 11 shows the molecular structure of compound D.

FIG. 12 shows the molecular structure of compound E

FIG. 13 shows the molecular structure of compound F.

Table 1 shows chemical structural formulae of compounds A, B, C, D, E,F, G and H.

Table 2 shows crystallographic data, parameters and refinements ofcompounds A, B, C, D, E and F.

Table 3 shows structure-antitussive activity relationship of fivenaturally occurring Stemona alkaloids. Data are expressed as mean ±SEM.*P<0.05 and ***P<0.001 compared with the vehicle control.

Table 4 shows structure-antitussive activity relationship of threesynthetic Stemona alkaloids and compound D. Data are expressed as mean±SEM. *P<0.05 and ***P<0.001 compared with the vehicle control.

Table 5 shows results of effects of different antagonists on theantitussive activity produced by compound A. Data are expressed as mean±SEM.

Table 6 shows results of radiolabeled ligand binding assays of compoundA. ± indicates slight but not significant inhibition on ligand binding.

DETAILED DESCRIPTION OF THE INVENTION

I. General

The present invention provides antitussive compounds such as Stemonaalkaloids, plant extracts, compositions, methods of preparation andmethods of using the same. Advantageously, the antitussive compounds,plant extracts, and compositions are efficacious to treat coughing andare free of side-effects. The antitussive compounds, plant extracts, andcompositions effectively reduce and or suppress coughing. As usedherein, the term “antitussive activity,” refers to the reduction orsuppression of coughing.

A. Compounds

The present invention provides antitussive compounds, such as Stemonaalkaloids useful in reducing or suppressing coughing. As will beapparent to one of skill in the art, certain compounds of the presentinvention possess asymmetric carbon atoms (chiral centers) or doublebonds; the racemates, diastereomers, enantiomers, geometric isomers andindividual isomers are all intended to be encompassed within the scopeof the present invention.

In one aspect, the antitussive compounds have Formula I:

wherein R¹, R², R³, R⁴, R⁵ and R⁶ have been previously defined. FIG. 1Ashows the chemical structural formulae of four isolated novel Stemonaalkaloids, compounds B, C, D and E. FIG. 1B shows the chemicalstructural formulae of three synthetic novel Stemona related alkaloids,compounds F, G, and H. FIG. 1C shows the chemical structural formula ofthe isolated known alkaloid, neotuberostemonine (compound A). Inparticularly preferred embodiments, the alkaloids of the presentinvention are represented by the chemical structural formulae ofcompounds A, B, C, D, E, F, G, or H, as set forth in Table I.

TABLE 1 Chemical structural formulae of compounds A, B, C, D, E, F, Gand H

I Comp R¹ R² R³ R⁴ R⁵ R⁶ A

B

C

D H

E

F H

—CH₂—OH

G H

—COOH

H H

The compounds of the present invention include both natural products andsynthetic analogs. In certain preferred aspect, the natural products ofthe present invention have the following formula:

wherein R¹ is selected from a hydrogen and anα(S)-methyl-γ(S)-butyroylactonyl; R² is selected from a β-orientedhydrogen and an α-oriented hydrogen; and R³ is selected from aβ-oriented hydrogen and an α-oriented hydrogen. In one preferred aspect,R¹ is an α(S)-methyl-γ(S)-butyroylactonyl, R² is an α-oriented hydrogenand R³ is an α-oriented hydrogen, which is also referred to herein ascompound B. In another preferred aspect, R¹ is anα(S)-methyl-γ(S)-butyroylactonyl, R² is an α-oriented hydrogen and R³ isa β-oriented hydrogen, which is also referred to herein as compound C.In still another preferred aspect, R¹ is a hydrogen, R² is β-orientedhydrogen, and R³ is β-oriented hydrogen, which is also referred toherein as compound D. In still another aspect, the compound of FormulaIa has Formula Ib, which is also referred to herein as compound E.Compound E has the formula:

In addition to the natural products, the present invention also includessynthetic analogs. In certain preferred aspects, the natural productscan be derivatized to generate compounds of Formula Ic:

wherein: R⁴ is a hydroxyl; and R⁵ is selected from a hydroxymethyl and acarboxyl; or alternatively, R⁴ and R⁵ together with the carbons to whichthey are attached, join to form a substituted furane ring. In onepreferred aspect, R⁴ is a hydroxyl, and R⁵ is a hydroxymethyl, which isalso referred to herein as compound F. In another preferred aspect, R⁴is a hydroxyl and R⁵ is a carboxyl, which is also referred to herein ascompound G. In still another preferred aspect, the compound has theformula

which is also referred to herein as compound H.

B. Methods of Making

As discussed above, the compounds of the present invention include bothnatural products and synthetic analogs. The naturally occurringcompounds, such as compounds of Formula Ia and Ib, can be obtained froma Stemonaceae family plant extract, such as S. tuberosa. These naturallyoccurring compounds can be obtained from a total alkaloid extract. Atotal alkaloid extract can be produced using methods according to thepresent invention.

As such, the present invention provides a method for preparing a totalalkaloid extract having antitussive activity from a Stemonaceae familyplant, the method comprising: (a) contacting a Stemonaceae family plantsample with an alcohol, such as ethanol to form a liquor; (b)evaporating the alcohol in the liquor to form a syrup; (c) adjusting thesyrup to an acid pH to form a supernatant fraction; (d) adjusting thesupernatant fraction to a basic pH, and then extracting with an organicsolvent, such as diethyl ether, to form an organic solution. The organicsolution can thereafter be evaporated to dryness to form the totalalkaloid extract, and can be further separated using for example, columnchromatography, to obtain the pure alkaloids of interest.

In one embodiment, a silica gel column is eluted successively with adiscontinuous gradient solvent system to yield fractions containingalkaloids of the present invention. The fractions can be monitored usingwell-known techniques to those of skill in the art. The fractions canthereafter be crystallized to yield compounds B, C, D, and E.

In certain aspect, the naturally occurring alkaloids of the presentinvention possess a fused lactone ring. These lactone ringfunctionalities can be derivatized or reduced to generate additionalcompounds of the present invention. In one preferred aspect, compound Dis reduced with lithium aluminum hydride to yield compounds F or H. Inanother aspect, the lactone ring of compound D is hydrolyzed to yieldcompound G. As such, in certain aspects, the present invention providesmethods of making compounds of Formula Ic and Id.

C. Plant Extracts

In another embodiment, the present invention provides a plant extracthaving antitussive activity. In some preferred embodiments, the plantextract is a Stemonaceae family plant extract having antitussiveactivity, wherein the Stemonaceae family plant extract inhibits a coughin a subject. In certain aspects, the genus belonging to the Stemonaceaefamily is for example, Stemona, Croomia, or Stichoneuron. Preferably,the genus is Stemona or Croomia. The species of the Stemona or Croomiagenus can be for example, S. collinsae, S. japonica, S. mairei, S.parviflora, S. sessilifolia, S. tuberosa, C. japonica, and C.heterosepala. (C. represents Croomia). Preferably, the Stemona speciesis Stemona tuberosa. As discussed above, the plant extract can be anaqueous extract or a total alkaloid extract. The plant extract cancomprise natural products, such as compounds of Formula Ia, Ib, andcompound A described in Table 1 and mixtures thereof.

In one embodiment, the present invention provides a method for preparinga total alkaloid extract having antitussive activity from a Stemonaceaefamily plant, comprising: (a) contacting a Stemonaceae family plantsample with an alcohol to form a liquor; (b) evaporating the alcohol inthe liquor to form a syrup; (c) adjusting the syrup to an acid pH toform a supernatant fraction; (d) adjusting the supernatant fraction to abasic pH, and then extracting with an organic solvent to form an organicsolution; and (e) evaporating the organic solvent to dryness to form atotal alkaloid extract having antitussive activity.

In another embodiment, the present invention provides a method forpreparing an aqueous extract having antitussive activity from aStemonaceae family plant, comprising: (a) contacting a Stemonaceaefamily plant sample with an aqueous solvent to form a liquor; and (b)evaporating the liquor to dryness to form an aqueous extract havingantitussive activity.

D. Compositions

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising the compounds of the present invention or anextract and a pharmaceutically acceptable carrier. Pharmaceuticallyacceptable carriers are determined in part by the particular compositionbeing administered, as well as by the particular method used toadminister the composition. Accordingly, there is a wide variety ofsuitable formulations of pharmaceutical compositions of the presentinvention. Formulations suitable for oral administration can consist of(a) liquid solutions, such as an effective amount of the alkaloiddissolved in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions.

Tablet forms can include one or more of lactose, sucrose, mannitol,sorbitol, calcium phosphates, corn starch, potato starch, tragacanth,microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide,croscarmellose sodium, talc, magnesium stearate, stearic acid, and otherexcipients, colorants, fillers, binders, diluents, buffering agents,moistening agents, preservatives, flavoring agents, dyes, disintegratingagents, and pharmaceutically compatible carriers. Lozenge forms cancomprise the active ingredient in a flavor, usually sucrose and acaciaor tragacanth, as well as pastilles comprising the active ingredient inan inert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art.

In a preferred embodiment, the compositions of the present invention areused in the treatment of coughs. In a preferred embodiment the inventionprovides a long-lasting cough composition. The dosages of the abovecompositions can vary depending on many factors such as thepharmacodynamic characteristics of the particular substance, and itsmode and route of administration; age, health, and weight of theindividual recipient; nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired.

The compositions of the present invention preferably contain suitablepharmaceutical carriers or diluents. Suitable pharmaceutical carriersand methods of preparing pharmaceutical dosage forms are described inRemington's Pharmaceutical Sciences, Mack Publishing Company, a standardreference text in this field. Suitable pharmaceutical diluents,excipients, or carriers suitable selected with respect to the intendedform of administration, that is, oral tablets, capsules, elixirs, syrupsand the like, consistent with conventional pharmaceutical practices. Thecompositions are preferably for oral delivery, more preferably in theform of a capsule or syrup, such as a cough syrup.

For oral administration in liquid form, the oral active substances canbe combined with any oral, non-toxic, pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water, and the like. Suitablebinders, lubricants, disintegrating agents, and coloring agents can alsobe incorporated into the dosage form if desired or necessary. Suitablebinders include starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth, or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes, and the like. Suitable lubricants used inthese dosage forms include sodium oleate, sodium stearate, magnesiumstearate, sodium benzoate, sodium acetate, sodium chloride, and thelike. Examples of disintegrators include starch, methyl cellulose, agar,bentonite, xanthan gum, and the like. Cough formulations generallyinclude (in addition to the active ingredients) sorbitol, saccharose,citric acid, flavoring and water.

For oral administration in the form of a table or capsule, the activesubstances can be combined with an oral, non-toxic, pharmaceuticallyacceptable, inert carrier such as lactose, starch, sucrose, glucose,methyl, cellulose, magnesium stearate, dicalcium phosphate, calciumsulfate, mannitol, sorbitol and the like. Gelatin capsules may containthe active substance and powdered carriers, such as lactose, starch,cellulose derivatives, magnesium stearate, stearic acid, and the like.Similar carriers and diluents may be used to make compressed tablets.Tablets and capsules can be manufactured as sustained release productsto provide for continuous release of active ingredients over a period oftime. Compressed tablets can be sugar coated or film coated to mask anyunpleasant taste and protect the tablet from the atmosphere, or entericcoated for selective disintegration in the gastrointestinal tract.Liquid dosage forms for oral administration may contain coloring andflavoring agents to increase subject acceptance.

Water, a suitable oil, saline, aqueous dextrose, and related sugarsolutions and glycols such as propylene glycol or polyethylene glycols,may be used as carriers for parenteral solutions. Such solutions alsopreferably contain a water soluble salt of the active ingredient,suitable stabilizing agents, and if necessary, buffer substances.Suitable stabilizing agents include antioxidizing agents such as sodiumbisulfate, sodium sulfite, or ascorbic acid, either alone or combined,citric acid and its salts and sodium EDTA. Parenteral solutions may alsocontain preservatives, such as benzalkonium chloride, methyl- orpropylparaben, and chlorobutanol.

The compounds of the invention may also be administered in the form ofliposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles, and multilamelar vesicles. Liposomes can be formedfrom a variety of phospholipids, such as cholesterol, stearylamine, orphosphatidylcholines. The compounds of the invention may also be coupledwith soluble polymers which are targetable drug carriers. Examples ofsuch polymers include polyvinylpyrrolidone, pyran copolymer,polyhydroxypropyl-methacrylamide-phenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. The substances may also be coupledto biodegradable polymers useful in achieving controlled release of adrug. Suitable polymers include polylactic acid, polyglycolic acid,copolymers of polylactic and polyglycolic acid, polyepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacylates, and crosslinked or amphipathicblock copolymers of hydrogels.

More than one compounds of the invention may be used in a composition.The compounds can be administered concurrently, separately orsequentially.

E. Methods of Using

The present invention provides a method for suppressing cough in amammal comprising the step of administering a pharmaceutically effectiveamount of an alkaloid represented by the chemical structural formulae ofcompound A, B, C, D, E, F, G, H or combinations and mixtures thereof. Inanother aspect, the present invention provides a method for reducingcoughing in a subject, comprising: administering a pharmaceuticallyeffective amount of a Stemonaceae family plant extract havingantitussive activity, wherein the Stemonaceae family plant extractinhibits a cough in a subject.

The dose administered to a subject, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the subject over time. The dose will be determined by thecondition of the subject. The size of the dose also will be determinedby the existence, nature, and extent of any adverse side effects thataccompany the administration to a particular subject. In determining theeffective amount of alkaloid to be administered to achieve antitussiveeffect, standard dosing regimens may be used as known in the art.Karlsson et al., Pulmonary Pharmacology and Therapeutics (1999)12:215-238.

In another aspect, the present invention features a method for preparingan isolated alkaloid for administering to an animal to reduce coughing,comprising: (a) providing a plant sample; (b) contacting the plantsample with an alcohol so that soluble plant chemical compositions areextracted from the plant sample into the alcohol to form a liquor; (c)evaporating the alcohol in the liquor to form a syrup; (d) adjusting thesyrup to an acid pH and separating out particulate matter in the syrupto form a supernatant fraction; (e) adjusting the supernatant fractionto a basic pH and extracting the soluble plant chemical compositionswith at least one solvent to form an organic solution; (f) evaporatingthe organic solution to dryness to form a total alkaloid extract; (h)purifying, separating and crystallizing the total alkaloid extract toform an isolated alkaloid; and (i) administering the isolated alkaloidto an animal having coughing episodes, thereby, reducing or suppressingthe coughing episodes.

As set forth in FIG. 2, an aqueous extract having antitussive activitysignificantly inhibited citric acid-induced cough by about 50% at a highdose of 3 g/kg and increased cough latency along with increase indosage. FIGS. 2A-B illustrate the antitussive effects of the aqueousextract derived from Stemona tuberosa. FIG. 2A shows the percentage ofcough episodes of the control and FIG. 2B shows the ratio of coughlatency between the extract and the control.

At a dose of 150 mg/kg, the total alkaloid extract significantly reducedthe number of coughs (see, FIG. 3). The results demonstrated that totalalkaloid extract derived from Stemona tuberosa is effective againstcough. Furthermore, the results also indicated that the antitussiveactivity produced by the total alkaloid extract is very potent and thus,Stemona alkaloids present in such extract produce the antitussiveactivity.

In another embodiment, a method is provided for screening for asubstance that inhibits coughing activity. For example, a guinea pig ischallenged with 0.5 M citric acid. Those producing more than 10 coughepisodes during a first citric acid challenge are selected to besensitive to citric acid induction and are used for further antitussivetests. The number of coughs and the cough latency in the first challengeanimal are recorded as the basal level control. After 48 hours ofrecovery, the sensitive guinea pigs are selected and are randomlydivided into different group, and then pretreated with eitherintraperitoneal or oral administration. For the intraperitonealadministration, the administration can be of a aqueous extract (0.3-3g/kg), a total alkaloid extract (25-150 mg/kg), an alkaloid, for examplecompound A (10, 30, 50, and 100 mg/kg), or compounds B, C, D, F, G, H(133 μmol/kg), for 30 min, or of an extract suspected of havingantitussive activity. For the oral administration, the administrationcan be of compound A or E (400 μmol/kg) for 60 min, or of an extractsuspected of having antitussive activity. A second citric acid exposureis then conducted. Antitussive activity is evaluated in each treatedanimal as the reduction of coughs and the increase of cough latency, ascompared to the basal level control.

The potency of antitussive activity produced by each alkaloid tested wascompared statistically using GraphPad PRISM software (Version 3.00 forWindows 95 and NT, GraphPad Software Inc., San Diego, Calif., USA).Parametric tests were used where possible, since the preliminary studyindicated that the data based on cough episode and latency pass in thenormality test. To compare the potency between two groups with differenttreatments, a paired or unpaired T-test was performed, and the groupsselected for matched or unmatched data. For comparison among differentgroups, a one-way analysis of variance (ANOVA) followed by aBonfferoni's test was performed. The probability (P) value of less than0.05 obtained from the results of the comparison of different groups wasconsidered to be statistically significant different. Based on thestatistical results of these tests, the potency of all the testedalkaloids was determined and the relationship between structure andactivity was defined.

II. EXAMPLES

Materials

1. Instrumentation

Melting points were measured using a Fisher Scientific instrument andwere uncorrected. Optical rotations were recorded on a Perkin-Elmer 341Polarimeter in MeOH solution. IR spectra were recorded on a NicoletImpact 420 FT-IR spectrometer. EI-MS was performed on a Finnegan MAT GCQwith a direct loop injection, while ESI-MS was recorded on a FinneganMAT TSQ 7000 instrument. NMR spectra (¹H and ¹³C NMR) were recorded oneither a 500 MHz or 300 MHz Bruker spectrometer in CDCl₃. The chemicalshifts are reported in δ (ppm) with TMS as an internal standard andcoupling constants (J) are given in Hz. X-ray diffractions for compoundsB, C and D were conducted on a Bruker SMART CCD diffractometer and thosefor compounds A, E and F were conducted on a Bruker P4 diffractometer.Merck silica gel (60 F₂₅₄) precoated on an aluminum sheet was used forthin layer chromatography (TLC), on which the spots were detected byspraying with Dragendorff reagent. Merck silica gel (70-230 mesh) wasused for column chromatography.

2. Plant Material

The dried root tuber of Stemona tuberosa used in the above experimentswas purchased from a Chinese herbal store in Hong Kong, and verified asStemona tuberosa. A voucher specimen (#992300) of this plant materialhas been deposited in the museum of Institute of Chinese Medicine, TheChinese University of Hong Kong, Hong Kong.

3. Animals

Male adult Dunkin-Hartley guinea pigs (body weight 300-500 g), suppliedby the Laboratory Animal Services Center, the Chinese University of HongKong, were used in this invention. The guinea pigs were maintained inclimatized colony-rooms (temperature 21±1° C.; humidity 60%) on anatural light/dark cycle with access to standard food and water.

Example 1

Production of Aqueous Extract

Dried root tubers of Stemona tuberosa (10 g) were chopped into smallpieces and extracted with distilled water under reflux for 2 hr. Afterfiltration the water solution was evaporated to dryness to yield aaqueous extract (3.5 g).

Example 2

Production of Total Alkaloid Extract

To obtain a total alkaloid extract, 6 kg of dried root tubers of Stemonatuberosa was chopped into small pieces and refluxed with 95% ethanol for2 hr. The warm extract liquor was poured out and allowed to standovernight at 10° C. Thereafter, the liquor was filtered, and thefiltrate evaporated under reduced pressure to obtain a syrup. The syrupwas acidified with diluted hydrochloride solution (4%) and centrifugedat 3000 RPM, 5° C., for 40 min. The supernatant was basified withaqueous ammonium hydroxide to pH 9 and sequentially extracted withdiethyl ether and chloroform, respectively. The combined organicsolution was then evaporated to dryness resulting in a total alkaloidextract (24 g).

Example 3

Isolation of Five Stemona Alkaloids (Compounds A-E)

The total alkaloid extract (24 g) of Example 2 was dissolved in diethylether with refluxing, then left overnight at room temperature, to give alight yellow precipitate, the crude compound A (3.4 g). The crudecompound A was further crystallized with ethanol resulting in a purealkaloid (Compound A, 2.5 g). The combined mother liquor was evaporatedto dryness, to form a mixture of alkaloids. The mixture of alkaloids wasthen subjected to silica gel column chromatography, and elutedsuccessively with a discontinuous gradient solvent of CHCl₃:MeOH:NH₄OH(98:2:0.05), (96:4:0.05) and (92:8:0.05), respectively. The eluant wasmonitored by Thin Layer Chromatography (TLC) and pooled into fourfractions 1-4. Fraction 1 (0.9 g) eluted with CHCl₃:MeOH:NH₄OH(98:2:0.05) was further purified by silica gel column chromatographywith hexane-EtOAc (70:30) elution to yield compound E(Eipbisdehydrotuberostemonine J, 380 mg). Fraction 2 (0.8 g) eluted withCHCl₃:MeOH:NH₄OH (98:2:0.05) was further purified by silica gel columnchromatography with hexane-EtOAc (60:40) elution, resulting in CompoundB (Tuberostemonine J, 245 mg). Fraction 3 (2.3 g) eluted withCHCl₃:MeOH:NH₄OH (96:4:0.05) was further fractionated by silica gelcolumn chromatography with hexane-EtOAc (50:50) elution, resulting inCompound C (Tuberostemonine H, 470 mg) and Compound A (98 mg),respectively. Fraction 4 (3.1 g) eluted with CHCl₃:MeOH:NH₄OH(92:8:0.05) was fractionated by silica gel column chromatography withhexane-EtOAc (35:65) elution to obtain Compound D (Neostenine, 600 mg).

The characteristics of each compound are given below.

Compound A (Neotuberostemonine): A colorless needle crystallized fromhexane/EtOAc. mp: 159.5-161° C.; [α]_(D) ²⁰=+83° (c, 0.1; MeOH); IRν_(max) ^(KBr) cm⁻¹: 1762, 1456, 1167, 1015; ESI-MS m/z (% intensity):376 [M+H]⁺ (63); EI-MS m/z (% intensity): 375 [M]⁺ (5), 276 [M-C₅H₇O₂]⁺(100). ¹H NMR (300 MHz, CDCl₃) δ: 0.96 (3H, t, J=7.3 Hz, H-17), 1.20(3H, d, J=7.1 Hz, H-15), 1.22 (3H, d, J=7.0 Hz, H-22), 1.3-2.0 (14H,H-1, 2H-2, 2H-6, 2H-7, 2H-8, H-9, H-10, 2H-16, H-19), 2.05 (1H, ddd,J=15.0, 3.3, 6.6 Hz, H-12), 2.34 (1H, ddd, J=5.4, 13.3, 15.2 Hz, H-19),2.58 (1H, ddq, J=5.3, 7.0, 12.1 Hz, H-20), 2.84 (1H, dq, J=6.6, 6.9 Hz,H-13), 2.92 and 3.03 (each 1H, m, 2H-5), 3.16 (1H, dd, J=3.8, 3.9 Hz,H-9a), 3.29 (1H, dd, J=7.7, 14.0 Hz, H-3), 4.36 (1H, ddd, J=5.4, 7.7,11.2 Hz, H-18), 4.49 (1H, dd, J=3.3, 3.0 Hz, H-11). The X-raydiffraction data are summarized in Table 2 and FIG. 8.

Compound B (Tuberostemonine J): A colorless prism crystallized fromhexane/EtOAc. mp: 180-182° C.; [α]_(D) ²⁰=+36.4° (c, 0.1; MeOH); EI-MSm/z (% intensity): 375 [M]⁺ (1.5), 276 [M-C₅H₇O₂]⁺ (100). ¹H NMR (500MHz, CDCl₃) δ: 1.01(3H, t, J=7.5 Hz, H-17), 1.18 (3H, d, J=7.5 Hz,H-15), 1.22 (3H, d, J=7.5 Hz, H-22), 1.40-2.10 (15H, H-1, 2H-2, 2H-6,2H-7, 2H-8, H-9, H-10, H-12, 2H-16, H-19), 2.25 (1H, m, H-19), 2.50 (1H,m, H-20), 2.74 (1H, m, H-13), 2.74 and 2.98 (each 1H, m, 2H-5), 3.02(2H, m, H-3 and H-9a), 4.19 (1H, br, H-11), 4.37 (1H, m, H-18); ¹³C NMR(125 MHz, CDCl₃) δ: 11.60 (C-17), 12.90 (C-15), 14.80 (C-22), 25.45(C-16), 29.45 (C-7), 30.62 (C-6), 32.40 (C-2), 33.27 (C-8), 34.30(C-19), 34.52 (C-9), 34.77 (C-10), 38.38 (C-12), 41.11 (C-20), 45.05(C-13), 45.80 (C-1), 50.09 (C-5), 64.59 (C-3), 66.28 (C-9a), 80.18(C-11), 81.28 (C-18), 179.18 (C-14), 179.26 (C-21). The X-raydiffraction data are summarized in Table 2 and FIG. 9.

Compound C (Tuberostemonine H): A colorless needle crystallized formhexane/EtOAc. mp: 183-185° C.; [α]_(D) ²⁰=+77.6°(c, 0.1; MeOH); IRν_(max) ^(KBr) cm⁻¹: 1769, 1454, 1174, 1016; EI-MS m/z (% intensity):375 [M]⁺ (0.9), 276 [M-C₅H₇O₂]⁺ (100). ¹H NMR (500 MHz, CDCl₃) δ: 1.00(3H, t, J=7.2 Hz, H-17), 1.18 (3H, d, J=7.2 Hz, H-15), 1.22 (3H, d,J=7.2 Hz, H-22), 1.3-2.0 (15H, H-1, 2H-2, 2H-6, 2H-7, 2H-8, H-9, H-10,H-12, 2H-16, H-19), 2.35 (1H, m, H-19), 2.45 (1H, m, H-20), 2.61 (1H, m,H-13), 2.78 and 2.84 (each 1H, m, 2H-5), 3.01 (1H, m, H-9a), 3.20 (1H,m, H-3), 4.37 (1H, ddd, J=4.6, 5.9, 10.5 Hz, H-18), 4.57 (1H, d, J=3.6Hz, H-11); ¹³C NMR (125 MHz, CDCl₃) δ: 11.61 (C-15), 11.88 (C-17), 15.05(C-22), 21.17 (C-16), 24.10 (C-7), 27.10 (C-8), 27.34 (C-6), 31.15(C-2), 33.42 (C-19), 35.31 (C-10), 41.10 (C-9), 41.90 (C-20), 44.14(C-12), 44.84 (C-1), 47.22 (C-13), 54.70 (C-5), 67.46 (C-9a), 77.97(C-3), 79.24 (C-18), 80.67 (C-11), 179.10 (C-21), 179.45 (C-14). TheX-ray diffraction data are summarized in Table 2 and FIG. 10.

Compound D (Neostenine): A colorless prism crystallized fromhexane/EtOAc. mp: 90-92° C.; [α]_(D) ²⁰=+73.6° (c, 0.1; MeOH); EI-MS m/z(% intensity): 277 [M]⁺ (74), 276 [M−H]⁺ (100), 233 (32), 204 (76),191(67). ¹H NMR (500 MHz, CDCl₃) δ: 0.97 (3H, t, J=7.5 Hz, H-17), 1.20(3H, d, J=7.2 Hz, H-15), 1.3-2.0 (14H, H-1, 2H-2, 2H-6, 2H-7, 2H-8, H-9,H-10, H-12, and 2H-16), 2.27 (1H, m, H-13), 2.45 (2H, m, H-3), 2.81 and2.89 (each 1H, m, 2H-5), 3.22 (1H, m, H-9a), 4.50 (1H, br, H-11); ¹³CNMR (125 MHz, CDCl₃) δ: 10.67 (q, C-17), 11.84 (q, C-15), 21.63 (t,C-16), 21.70 (t, C-7), 28.67 (t, C-8), 28.86 (t, C-6), 30.65 (t, C-2),34.79 (t, C-9), 37.81 (d, C-10), 37.97 (d, C-12), 43.04 (d, C-13), 43.38(d, C-1), 56.28 (t, C-5), 56.44 (t, C-3), 71.52 (d, C-9a), 79.87 (d,C-11), 180.24 (s, C-14). The X-ray diffraction data are summarized inTable 2 and FIG. 11.

Compound E (Epibisdehydrotuberostemonine J): A colorless prismcrystallized from hexane/EtOAc. mp: 186-188° C.; [C]_(D) ²⁰=−16.1° (c,0.1; MeOH); IR ν_(max) ^(KBr) cm⁻¹: 2932, 2864, 1763, 1454, 1158, 997,923; EI-MS m/z (% intensity): 371 [M]⁺ (68), 327 (28), 298 (100), 272[M-C₅H₇O₂]⁺ (71). ¹H NMR (500 MHz, CDCl₃) δ: 1.06 (3H, t, J=7.0 Hz,H-17), 1.35 (3H, d, J=7.0 Hz, H-22), 1.37 (3H, d, J=7.0 Hz, H-15), 1.47(1H, t, J=11.5 Hz, H-10), 1.80 (2H, m, H-16), 1.95 (2H, m, H-8), 2.06and 1.16 (each 1H, m, 2H-6), 2.08 and 1.47 (each 1H, m, 2H-7), 2.07 (1H,d, J=11 Hz, H-19), 2.7-2.9 (3H, m, H-13, H-19, H-20), 3.01 (1H, t, J=7.0Hz, H-9), 3.56 (1H, dd, J=5.0, 5.8 Hz, H-12), 3.79 (1H, dd, J=13.3, 14.0Hz, H-5), 4.24 (1H, dd, J=5.0, 14.0 Hz, H-5), 4.67 (1H, d, J=4.0 Hz,H-11), 5.36 (1H, dd, J=5.0, 11.0 Hz, H-18), 5.99 (1H, s, H-2); ¹³C NMR(125 MHz, CDCl₃) δ: 11.39 (C-17), 11.88 (C-15), 14.97 (C-22), 23.25(C-16), 28.58 (C-6), 28.88 (C-7), 30.97 (C-19), 34.87 (C-10), 35.04(C-8), 36.10 (C-9), 39.57 (C-12), 41.80 (C-13), 41.94 (C-20), 44.85(C-5), 71.70 (C-18), 80.89 (C-11), 107.13 (C-2), 108.59 (C-1), 126.60(C-9a), 137.58 (C-3), 178.87 (C-14), 178.87 (C-21). The X-raydiffraction data are summarized in Table 2 and FIG. 12.

TABLE 2 Crystallographic data, parameters, and refinements of compoundsA, B, C, D, E and F Parameter Compound Alkaloid A B C D E F Size (mm)0.30 × 0.40 × 0.75 0.38 × 0.36 × 0.77 0.22 × 0.34 × 0.65 0.26 × 0.28 ×0.82 0.12 × 0.40 × 0.72 0.38 × 0.30 × 0.8 Chemical C₂₂H₃₃NO₄ C₂₂H₃₃NO₄C₂₂H₃₃NO₄ C₁₇H₂₇NO₂ C₂₂H₂₉NO₄ C₁₇H₃₁NO₂ formula Formula 375.49 375.49375.49 277.40 371.46 281.43 weight Crystal Orthorhombic OrthorhombicOrthorhombic Orthorhombic Monoclinic Orthorhombic system Space groupP2(1)2(1)2(1) P2(1)2(1)2(1) P2(1)2(1)2(1) P2(1)2(1)2(1) P2(1)P2(1)2(1)2(1) Unit cell a = 6.459(2) a = a = 5.5337(5) a = 5.8368(4) a =a = 7.9979(14) dimension 9.0115(11) 6.3596(19) b =14.228(3) b =10.612(4)b = b = 9.6805(7) b = 18.495(3) b = 18.495(3) 19.0275(18) c = 23.033(3)c = 22.074(3) c = c = 27.789(2) c = c = 21.515(4) 20.0554(19) 8.3875(15)β = 92.521(18) Volume, Å³ 2116.8(9) 2110.9 2111.7 1570.1(2) 985.6(4)1640.7(5) Z 4 4 4 4 2 4 Density 1.178 1.182 1.162 1.173 1.252 1.139(calculated) mg/m³ Reflections 2891 3109 12774 7580 2449 9269 measuredIndependent 2690 2902 4138 2277 1914 2909 Observed 1661 1750 1805 17441383 1872 reflections Parameters 246 246 244 182 246 183 Goodness of1.060 1.017 0.999 0.986 1.048 0.999 fit on F² Final R 0.050 0.058 0.0690.038 0.045 0.046 indices [I > 4σ] R indices 0.098 0.108 0.181 0.0580.077 0.092 (all data)

Example 4

Synthesis of Analogs of Compound D: Compounds F, G. and H

4.1 Synthesis of Compound F

A mixture of Compound D (250 mg) and lithium aluminum hydride (120 mg)was heated under reflux in tetrahydrofuran with stirring for 3 hrs.After cooling, a few drops of water was added to the mixture to destroythe excess lithium aluminum hydride. The mixture was filtered and thefiltrate was evaporated to dryness under reduced pressure at 40 ° C. toobtain a white residue. The residue was dissolved in a 4% hydrochloridesolution and then extracted with diethyl ether to remove non-alkaloidcompounds. The aqueous layer was separated out and basified with 25%ammonium hydroxide, and then extracted with diethyl ether. The diethylether layer was washed with water and evaporated to yield dried powders,which were crystallized from hexane-EtOAc to give prisms (165 mg). Theresulting synthetic compound was named Neostenine-diol. Thecharacteristics of Neostenine-diol are as follows.

Fine prismatic crystals from hexane-EtOAc; mp 130-132° C.; ESI-MS m/z (%intensity): 282 [M+H]⁺ (100). ¹H NMR (300 MHz, CDCl₃) δ: 0.93 (3H, t,J=7.5 Hz, H-17), 1.04 (3H, d, J=7.5 Hz, H-15), 2.4-2.7 (2H, m, H-3),2.9-3.1 (2H, m, H-5), 3.34 (1H, m, H-9a), 3.61 (2H, m, H-14), 3.90 (1H,m, H-11); ¹³C NMR (75.5 MHz, CDCl₃) δ: 12.18 (C-17), 18.90 (C-15), 21.58(C-7), 22.26 (C-16), 28.09 (C-2), 29.59 (C-8), 30.03 (C-6), 35.59(C-10), 38.58 (C-12), 39.45 (C-9), 43.83 (C-1), 44.99 (C-13), 56.87(C-3), 58.91 (C-5), 65.49 (C-14), 71.43 (C-9a), 73.04 (C-11). The X-raydiffraction data are summarized in Table 2 and FIG. 13.

4.2 Synthesis of Compound G

Compound D (148 mg) was dissolved in 2 ml of 4% sodium hydroxidesolution and heated with stirring on water both at 60° C. for 1.5 hrs.The suspended mixture changed into a clear solution. The solution wasthen surrounded by an ice bath, and adjusted gradually with 2%hydrochloride solution to pH 8, while stirring. Cooled distilled waterwas added into the resulting solution to make the final volume of 20 mlwith a final concentration of 26.67 mM of the product, compound F. Theresulting solution was stored at 0-5° C. This compound was namedNeostenine-acid.

The ESI-MS spectrum of compound G showed only one compound with amolecular weight of 296 [M+H]⁺ (100).

4.3 Synthesis of Compound H

A mixture of compound D (500 mg) and lithium aluminum hydride (250 mg)was refluxed with tetrahydrofuran for 4 hr. After cooling the mixture, afew drops of H₂O were added to destroy the excess lithium aluminumhydride. The mixture was then filtered and the filtrate evaporated todryness, forming a residue. The residue was dissolved in a 4% hydrogenchloride solution and then extracted with diethyl ether to removenon-alkaloid compounds. The aqueous layer was separated out and basifiedwith 25% NH₄OH, and then extracted with diethyl ether. The diethyl ethersolution was evaporated to dryness. The residue was heated with 20 ml of10% H₂SO₄ in a water bath for 5 hr, and the acidic solution wasextracted with diethyl ether, and then basified with K₂CO₃. Theresulting basic solution was extracted with diethyl ether. The organiclayer was washed with water, dried with K₂CO₃, and evaporated todryness, resulting in an oil. The oil was further purified by silica gelcolumn chromatography and eluted with a mixture of CHCl₃:EtOAc:MeOH(7:1:2) to obtain a colorless oil that was named Neostenine-ether (245mg). The characteristics of this compound are as follows.

ESI-MS m/z (% intensity): 264 [M+H]⁺ (100). ¹H NMR (300 CDCl₃) δ: 0.93(3H, t, J=7.5 Hz, H-17), 0.98 (3H, d, J=7.5, H-15), 3.22 (1H, m, H-9a),3.45 (1H, dd, J=8.1, 10.2 Hz, H-14), 3.87 (1H, dd, J=8.1, 8.1 Hz, H-14),4.1 (1H, m, H-11); ¹³C NMR (75.5 MHz, CDCl₃) δ: 12.13 (C-17), 12.97(C-15), 21.93 (C-16), 22.19 (C-7), 29.31 (C-2), 29.49 (C-8), 31.31(C-6), 35.09 (C-10), 37.03 (C-12), 38.69 (C-9), 38.81 (C-13), 42.89(C-1), 56.93 (C-3), 57.13 (C-5), 72.41 (C-14), 72.64 (C-9a), 80.06(C-11).

Example 5

Measurement of Antitussive Activity

The method previously described by Gallico et al. (1994) was adoptedwith the following modifications. Unrestrained guinea pigs were placedindividually in a hermetically sealed transparent Perspex chamber (25cm×12 cm×12 cm), and exposed to a nebulized aqueous solution of 0.5 Mcitric acid for 8 min. An ultrasonic nebulizer was used (OMRON NE-U12,Tokyo, Japan) that produced an aerosol with particles having a massmedian diameter of 1˜5 μm. Approximately 0.5 ml of 0.5 M citric acidsolution was nebulized per minute. During the exposure, the guinea pigswere continuously monitored by a trained observer. The episodes andlatency of coughing in 8 min were detected and counted by the observer.Cough sounds were recorded concurrently and amplified via a microphoneplaced in the chamber. The sound waves were analyzed by a personalcomputer by using Cool Edit 2000 software (Syntrillium, Phenix, USA).Therefore, cough counting was performed based on both acoustic data andhuman observations, and in this manner the reduction or inhibition ofcoughing (i.e., antitussive activity) was measured.

Example 6

Evaluation of Antitussive Activity of Extracts and Pure StemonaAlkaloids

The guinea pigs were first challenged with 0.5 M citric acid. Thoseproducing more than 10 cough episodes during the first citric acidchallenge were selected to be sensitive to citric acid induction andwere used for the further antitussive tests. The number of coughs andthe cough latency in the first challenge were recorded as the basallevel control. After 48 hours of recovery, the sensitive guinea pigswere selected and were randomly divided into different groups with atleast four guinea pigs in each group, and then pretreated withintraperitoneal administration of the aqueous extract (0.3-3 g/kg), thetotal alkaloid extract (25-150 mg/kg), compound A (10, 30, 50, and 100mg/kg), and compounds B, C, D, F, G, H (133 μmol/kg), for 30 min beforethe second citric acid exposure. Antitussive activity was evaluated ineach treated animal as the reduction of coughs and the increase of coughlatency, as compared to the basal level control. Furthermore, the sameantitussive test was also performed with oral administration ofcompounds A and E (400 μmol/kg). In addition, as a positive control, theantitussive activity of codeine (5, 10 and 30 mg/kg, i.p.) was alsodetermined in a parallel study.

Example 7

Antitussive Activities of Extracts and Compound A

As set forth in FIG. 2, an aqueous extract having antitussive activitysignificantly inhibited citric acid-induced cough by about 50% at a highdose of 3 g/kg and increased cough latency along with increase indosage. FIGS. 2A-B illustrate the antitussive effects of the aqueousextract derived from Stemona tuberosa. FIG. 2A shows the percentage ofcough episodes of the control and FIG. 2B shows the ratio of coughlatency between the extract and the control.

At a dose of 150 mg/kg, the total alkaloid extract significantly reducedthe number of coughs (see, FIG. 3). The results demonstrated that totalalkaloid extract derived from Stemona tuberosa is effective againstcough. Furthermore, the results also indicated that the antitussiveactivity produced by the total alkaloid extract is very potent and thus,Stemona alkaloids present in such extract produce the antitussiveactivity.

The antitussive activity of the pure Stemona alkaloids isolated fromStemona tuberosa, compound A, inhibited citric acid-induced cough in adose-dependent manner and also significantly increased the cough latencyat the doses of 50 and 100 mg/kg (see, FIG. 4). Furthermore, acomparative study was conducted using codeine, the commonly usedantitussive drug, as the positive control. The results showed that ED₅₀value for compound A and codeine were comparable.

Example 8

Structure-Antitussive Activity Relationship

The antitussive activities of the Stemona alkaloids, compounds B-H, wereexamined and their potencies were compared with that of compound A (see,FIG. 5). The 25 results demonstrated that both compounds A and Dsignificantly inhibited cough episodes in a dose-dependent manner.Compounds C, B, F and H exhibited marked reduction of cough episodes.Table 3 tabulates a structure-antitussive activity relationship of fivenaturally occurring Stemona alkaloids.

TABLE 3 Structure-antitussive activity relationship of five naturallyoccurring Stemona alkaloids

Substitution and % Cough Episode Relative Configuration No. of (Composedwith Alkaloid A/C B/C R Animals Vehicle Control) A cis cis

5  21.7 ± 8.9 (po)*  16.1 ± 7.3 (ip)*** B trans cis

5  76.3 ± 14.7 (ip) C trans trans

5  50.7 ± 16.6 (ip) D cis cis —H 9  29.9 ± 8.3 (ip)*** E — —

6 136.8 ± 25.6 (po)

Table 4 tabulates the structure-antitussive activity relationship ofthree synthetic Stemona alkaloids and compound D.

TABLE 4 Structure-antitussive activity relationship of three syntheticStemona alkaloids and compound D

% Cough Episode Substituted Group No. of (Composed with Alkaloid R₁   R₂Animals Vehicle Control) D

9  29.9 ± 8.3 (ip)*** F —CH₂—OH 5  62.2 ± 10.8 (ip) —OH G —COOH 4 104.5± 20.4 (ip) —OH H —CH₂—O— 6  54.4 ± 10.6 (ip)*

The rank order of antitussive potency was determined to be: compoundA=D>C≈F≈H≈B. The results suggested that the defined tricyclic ringsystem (rings A, B and C) (FIG. 1) is the primary key structurecontributing to the antitussive activity and all cis configurations atthree ring junctions are the optimal structure. Furthermore, thesubstituted groups at 3 position on ring A and at 11 and 12 positions onring C also affected the antitussive potency of these alkaloids and aα-methyl-γ-butyrolactone ring substitution in ring C produced thehighest potency amongst all tested Stemona alkaloids.

Example 9

In Vivo Test for Determining the Mechanism of Antitussive Activity.

Further studies were conducted for the delineation of the mechanisms ofantitussive activity produced by the representative Stemona alkaloid,compound A. Similarly, the sensitive animals were selected after thefirst citric acid challenge and allowed to recover for 24 hours. Duringthe second challenge, animals were treated with a subcutaneous injectionof different antagonists at different times prior to intraperitonealadministration of compound A (50 or 100 mg/kg). The antagonists examinedincluded SCH 50911 (GABA_(B) receptor antagonist) with a 40 minpretreatment at a dose of 10 mg/kg, WAY-100635 (5-HT_(1A) receptorantagonist) with a 60 min pretreatment at a dose of 1 mg/kg, andhaloperidol (Dopamine, D₂ receptor antagonist) with a 45 minpretreatment at a dose of 1 mg/kg. In addition, naloxone (nonselectiveopioid receptor antagonist) was administered subcutaneously at a dose of5 mg/kg at 20 min after intraperitoneal administration of compound A (30and 100 mg/kg). The effects of these antagonists on the antitussiveactivity of compound A were assessed by comparison of the changes incough episodes and cough latency between animal groups administratedwith compound A alone and concurrent administration of compound A andeach antagonist tested. Furthermore, in a parallel study, the sametreatment with naloxone was also conducted in the animals pretreatedwith intraperitoneal administration of codeine (30 mg/kg) for thepositive control. The results indicated that all antagonists tested didnot significantly affect the antitussive activity of Stemona alkaloids(FIG. 7, Table 5).

TABLE 5 Results of effects of different antagonists on the antitussiveactivity produced by compound A % Antitussive Activity of AntagonistReceptor Compound A Naloxone Opioid, (nonselective) 86.51 ± 11.57 SCH50911 GABA_(B) 83.25 ± 16.73 WAY-100635 5-HT_(1A) 100.50 ± 1.67 Haloperidol Dopamine (D₂) 115.21 ± 7.05 

Example 10

In Vitro Receptor Binding Assays for Determining the Mechanism ofAntitussive Activity

The representative Stemona alkaloid, compound A, was subjected to thefollowing primary receptor binding assays. The standard radiolabeledligand binding experiments were performed at the concentration of 10 μMin duplicate by Novascreen Biosciences Crop. (7170 Standard Drive,Hanover, Md. 21076, USA). The tested receptors included all currentlywell-known receptors involved in producing the antitussive activity, forexample, [³H]Deltorphin (δ₁-opioid receptor), [³H]Naltrindole (δ₂-opioidreceptor), [³H]DAMGO (μ-opioid receptor), [³H]U69593 (κ₁-opioidreceptor), [³1H]CGP 54626A (GABA_(B) receptor), [³H]8-OH-DPAT (5HT_(1A)receptor), and [3H]Glibenclamide (Dopamine D₂-receptor). In vitroreceptor binding assays (Table 6) also demonstrated thatneotuberostemonine did not show any affinity to all receptors examined.Therefore, the results obtained from both in vivo and in vitro studiesconfirmed that the antitussive activity of all five effective novelStemona alkaloids and neotuberostemonine are not involved in opiatereceptor pathways.

TABLE 6 Results of radiolabeled ligand binding assays of compound A %Inhibition of Ligand Receptor Source Ligand Binding (average) AffinityOpioid, δ₁ Rat forebrain [³H]Deltrophin 5.95 No Opioid, δ₂ Humanrecombinant [³H]Naltrindole 8.32 No Opioid, κ Guinea pig cerebellum[³H]U-69593 14.40 No Opioid, μ Rat forebrain [³H]DAMGO 7.37 No GABA_(B)Rat cortex [³H]CGP 54626A 8.06 No 5-HT_(1A) Bovine hippocampus[³H]8-OH-DPAT −18.42 No Dopamine, D₂ Rate striatum [³H]Glibenclamide33.39 ±

The examples and embodiments described herein are for illustrativepurposes only, and various modifications or changes thereof will besuggested to a person skilled in the art, and are included within thespirit and purview of this application and scope of the appended claims.All publications, patents, and patent applications cited herein arehereby incorporated by reference.

1. A compound having the formula:

wherein: R¹ is a member selected from the group consisting of hydrogenand α(S)-methyl-γ(S)-butyroylactonyl; R² is a member selected from thegroup consisting of β-oriented hydrogen and α-oriented hydrogen; R³ is amember selected from the group consisting of β-oriented hydrogen andα-oriented hydrogen; R⁴ is hydroxyl; R⁵ is a member selected from thegroup consisting of hydroxymethyl and carboxyl; and R⁶ is a memberselected from the group consisting of β-oriented hydrogen and α-orientedhydrogen; or alternatively, R² is an α-oriented hydrogen, and R⁴ and R⁵together with the carbons to which they are attached, join to form asubstituted γ(S)-butyroylactonyl or substituted furane ring; oralternatively, R¹ is hydrogen, and R⁴ and R⁵ together with the carbonsto which they are attached, join to form a substitutedγ(S)-butyroylactonyl or substituted ring; or alternatively, R⁴ and R⁵together with the carbons to which they are attached, join to form asubstituted γ(S)-butyroylactonyl, and R² and R⁶ are both absent and forma pyrrole ring, provided however that when R¹ isα(S)-methyl-γ(R)-butyroylactonyl, R³ is an α-oriented hydrogen.
 2. Thecompound of claim 1, wherein said compound has the formula

wherein: R¹ is hydrogen; R² is a member selected from the groupconsisting of β-oriented hydrogen and α-oriented-hydrogen; and R³ is amember selected from the group consisting of β-oriented hydrogen andα-oriented hydrogen.
 3. The compound of claim 1, wherein R¹ is anα(S)-methyl-γ(S)-butyroylactonyl; R² is an α-oriented hydrogen; R³ is anα-oriented hydrogen; and R⁴ and R⁵ together with the carbons to whichthey are attached, join to form a substituted γ(S)-butyroylactonyl. 4.The compound of claim 1, wherein R¹ is anα(S)-methyl-γ(S)-butyroylactonyl; R² is an α-oriented hydrogen; R³ is aβ-oriented hydrogen; and R⁴ and R⁵ together with the carbons to whichthey are attached, join to form a substituted γ(S)-butyroylactonyl. 5.The compound of claim 2, wherein R¹ is a hydrogen; R² is a β-orientedhydrogen; and R³ is a β-oriented hydrogen.
 6. The compound of claim 1,wherein said compound has the formula


7. The compound of claim 1, wherein said compound has the formula

wherein: R⁴ is a hydroxyl; and R⁵ is a member selected from the groupconsisting of hydroxymethyl and carboxyl; or alternatively, R⁴ and R⁵together with the carbons to which they are attached, join to form asubstituted furane ring.
 8. The compound of claim 7, wherein R⁴ is ahydroxyl; and R⁵ is a hydroxymethyl.
 9. The compound of claim 7, whereinR⁴ is a hydroxyl; and R⁵ is a carboxyl.
 10. The compound of claim 7,wherein said compound has the formula


11. A pharmaceutical composition, said composition comprising a compoundhaving the formula:

wherein: R¹ is a member selected from the group consisting of hydrogenand α(S)-methyl-γ(S)-butyroylactonyl; R² is a member selected from thegroup consisting of β-oriented hydrogen and α-oriented hydrogen; R³ is amember selected from the group consisting of β-oriented hydrogen andα-oriented hydrogen; R⁴ is hydroxyl; R⁵ is a member selected from thegroup consisting of hydroxymethyl and carboxyl; or alternatively, R⁴ andR⁵ together with the carbons to which they are attached, join to form asubstituted γ(S)-butyroylactonyl or substituted furane ring; and R⁶ is amember selected from the group consisting of β-oriented hydrogen andα-oriented hydrogen; or alternatively, R⁴ and R⁵ together with thecarbons to which they are attached, join to form a substitutedγ(S)-butyroylactonyl, and R² and R⁶ are both absent and form a pyrrolering, provided however that when R¹ is α(S)-methyl-γ(R)-butyroylactonyl,R³ is an α-oriented hydrogen; and a pharmaceutically acceptable carrier.12. The composition of claim 11, wherein said compound has the formula

wherein: R¹ is a member selected from the group consisting of hydrogenand α(S)-methyl-γ(S)-butyroylactonyl; R² is a member selected from thegroup consisting of β-oriented hydrogen and α-oriented hydrogen; and R³is a member selected from the group consisting of β-oriented hydrogenand α-oriented hydrogen.
 13. The composition of claim 12, wherein R¹ isan α(S)-methyl-γ(S)-butyroylactonyl; R² is an α-oriented hydrogen; andR³ is an α-oriented hydrogen.
 14. The composition of claim 12, whereinR¹ is an α(S)-methyl-γ(S)-butyroylactonyl; R² is an α-oriented hydrogen;and R³ is a β-oriented hydrogen.
 15. The composition of claim 12,wherein R¹ is a hydrogen; R² is a β-oriented hydrogen; and R³ is aβ-oriented hydrogen.
 16. The composition of claim 12, wherein R¹ is anα(S)-methyl-γ(S)-butyroylactonyl; R² is a β-oriented hydrogen; and R³ isa β-oriented hydrogen.
 17. The composition of claim 11, wherein saidcompound has the formula


18. The composition of claim 11, wherein said compound has the formula

wherein: R⁴ is a hydroxyl; and R⁵ is a member selected from the groupconsisting of hydroxymethyl and carboxyl; or alternatively, R⁴ and R⁵together with the carbons to which they are attached, join to form asubstituted furane ring.
 19. The composition of claim 18, wherein R⁴ isa hydroxyl; and R⁵ is a hydroxymethyl.
 20. The composition of claim 18,wherein R⁴ is a hydroxyl; and R⁵ is a carboxyl.
 21. The composition ofclaim 18, said compound has the formula


22. A method for reducing coughing in a subject, said method comprising:administering a pharmaceutically effective amount of a compound havingthe formula:

 wherein: R¹ is a member selected from the group consisting of hydrogenand α(S)-methyl-γ(S)-butyroylactonyl; R² is a member selected from thegroup consisting of β-oriented hydrogen and α-oriented hydrogen; R³ is amember selected from the group consisting of β-oriented hydrogen andα-oriented hydrogen; R⁴ is hydroxyl; R⁵ is a member selected from thegroup consisting of hydroxymethyl and carboxyl; or alternatively, R⁴ andR⁵ together with the carbons to which they are attached, join to form asubstituted γ(S)-butyroylactonyl or substituted furane ring; and R⁶ is amember selected from the group consisting of β-oriented hydrogen andα-oriented hydrogen; or alternatively, R⁴ and R⁵ together with thecarbons to which they are attached, join to form a substitutedγ(S)-butyroylactonyl, and R² and R⁶ are both absent and form a pyrrolering, provided however that when R¹ is α(S)-methyl-γ(R)-butyroylactonyl,R³ is an α-oriented hydrogen.
 23. The method of claim 22, wherein saidcompound has the formula

wherein: R¹ is a member selected from the group consisting of hydrogenand α(S)-methyl-γ(S)-butyroylactonyl; R² is a member selected from thegroup consisting of β-orientated hydrogen and α-oriented hydrogen; andR³ is a member selected from the group consisting of β-oriented hydrogenand α-oriented hydrogen.
 24. The method of claim 23, wherein R¹ is anα(S)-methyl-γ(S)-butyroylactonyl; R² is an α-oriented hydrogen; and R³is an α-oriented hydrogen.
 25. The method of claim 23, wherein R¹ is anα(S)-methyl-γ(S)-butyroylactonyl; R² is an α-oriented hydrogen; and R³is a β-oriented hydrogen.
 26. The method of claim 23, wherein R¹ is ahydrogen; R² is a β-oriented hydrogen; and R³ is a β-oriented hydrogen.27. The method of claim 23, wherein R¹ is anα(S)-methyl-γ(S)-butyroylactonyl; R² is a β-orientated hydrogen; and R³is a β-oriented hydrogen.
 28. The method of claim 22, wherein saidcompound has the formula


29. The method of claim 22, wherein said compound has the formula

wherein: R⁴ is hydroxyl; and R⁵ is a member selected from the groupconsisting of hydroxymethyl and carboxyl; or alternatively, R⁴ and R⁵and the carbons to which they are attached join, to form a substitutedfurane ring.
 30. The method of claim 29, wherein R⁴ is a hydroxyl; andR⁵ is a hydroxymethyl.
 31. The method of claim 29, wherein R⁴ is ahydroxyl; and R⁵ is a carboxyl.
 32. The method of claim 29, wherein saidcompound has the formula